Abstract 282: Knockdown of N-Myc and concurrent treatment with apigenin controlled growth of human malignant neuroblastoma cells having N-Myc amplification

Abstract

Malignant neuroblastomas, which mostly occur in children, are frequently associated with N-Myc amplification and poor prognosis. Conventional chemotherapeutic agents are not very effective for controlling malignant neuroblastomas having N-Myc amplification. Notably, N-Myc amplification has profound implication in sustained growth of malignant neuroblastomas because N-Myc amplification is associated with inhibition of differentiation and apoptosis and promotion of angiogenesis and invasion. We have hypothesized that knockdown of N-Myc using short hairpin RNA (shRNA) plasmid may increase anti-cancer efficacy of a flavonoid such as apigenin (APG) in human malignant neuroblastoma cells that are known to harbor N-Myc amplification. Our confocal laser scanning immunofluorescence microscopy following staining of the cells with FITC conjugated N-Myc antibody and DAPI nuclear stain showed that N-Myc was highly expressed in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines, moderately in IMR32 cell line, and slightly in SH-SY5Y cell line. Different levels of N-Myc expression in these cell lines were also confirmed by flow cytometric analyses. We selected SK-N-DZ and SK-N-BE2 cell lines, which showed high N-Myc expression, for further studies. We used semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting to determine the levels of knockdown of N-Myc in SK-N-DZ and SK-N-BE2 cell lines following treatments with nothing (control), scrambled shRNA, N-Myc shRNA, APG, and N-Myc shRNA plus APG. Combination therapy caused the highest levels of knockdown of N-Myc in both cell lines. Knockdown of N-Myc induced neuronal differentiation with changes in morphological features (increased cell length and neurite growth) and biochemical features including increased expression of neurofilament protein (NFL), neuron specific enolase (NSE), and e-cadherin and decreased expression of Notch-1, Id2, catalytic subunit of human telomerase reverse transcriptase (hTERT), and proliferating cell nuclear antigen (PCNA). Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing morphological and biochemical features of apoptosis. Combination therapy caused activation of caspase-8, cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, activation of calpain and caspase-3, and proteolysis of α-spectrin and ICAD. Combination therapy markedly decreased the migration of cells through matrigel and down regulated N-Myc driven survival factors (phospho-Akt and p65 NF-κB), angiogenic factors (VEGF and b-FGF), and invasive pathways (MMP-2 and MMP-9) in malignant neuroblastoma cells. Collectively, knockdown of N-Myc and concurrent APG treatment can be a promising therapeutic strategy for controlling growth of human malignant neuroblastoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 282. doi:1538-7445.AM2012-282